Báo cáo khoa học: " Packaging of viral RNAs in virions of adenoviruses" pptx

BioMed Central
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Virology Journal
Open Access
Research
Packaging of viral RNAs in virions of adenoviruses
Li Xing
1,3
and Suresh K Tikoo*
1,2
Address:
1
Vectored Vaccine Program, Vaccine & Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3,
Canada ,
2
Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3,
Canada and
3
Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada
Email: Li Xing - ; Suresh K Tikoo* -
* Corresponding author
Abstract
Earlier, we detected viral RNAs packaged in the porcine adenovirus (PAdV) -3 virions. Using
Southern blot analysis, we further demonstrated that the viral RNAs were predominantly packaged
in CsCl purified mature capsids (containing viral genome) than empty/intermediate capsids. Some
of the packaged viral RNAs appear to be polyadenylated. Real-time reverse transcription (RT)-PCR
analysis indicated that the copy number of the tested viral mRNAs encoding E1B
small
and fiber
proteins was less than one per full capsid. Moreover, detection of viral RNA packaged in CsCl

purified human adenovirus (HAdV) -5 virions indicates that the viral RNA packaging might be a
common phenomenon in members of Adenoviridae family. Further quantitative analysis of viral
protein, DNA, and RNA in CsCl purified mature and empty/intermediate capsids of recombinant
HAdV-5 expressing enhanced green fluorescent protein indicated that the traceable viral RNA
detected in empty/intermediate capsids seems associated with the presence of traceable viral
genomic DNA. Taken together, our data suggest that the viral RNAs may be passively packaged in
adenovirus virion during encapsidation of viral genomic DNA in cell nuclei. Thus, viral RNA
packaging may be a characteristic feature of adenoviral genomic DNA encapsidation.
Introduction
The phenomenon of encapsidation of viral RNAs was ini-
tially uncovered in members of Herpesviridae family [1-4].
These RNAs could be translated into proteins that would
function prior to de novo transcription from the viral
genome [1,2,5]. Alternatively, these viral RNAs might
facilitate organizing the structure of the tegument domain
through RNA-protein interactions during virion assembly
[6] as found in retroviruses [7,8]. Moreover, the studies
with human cytomegalovirus (HCMV) suggested that
both the viral and the cellular RNAs were nonspecifically
incorporated into the virions through interactions with
some virion proteins during budding [6]. However, stud-
ies with herpes simplex virus (HSV) -1 and Kaposi's sar-
coma-associated herpesvirus (KSHV) suggested that some
virion RNAs were specifically incorporated into the viri-
ons [1,4].
Adenoviruses are another family of DNA viruses that
infect a wide variety of mammals and birds [9]. Adenovi-
rus is non-enveloped containing a single, linear double-
stranded DNA of approximately 26–43 kb within an
icosahedral capsid of 70–100 nanometer in diameter

[10]. The assembly of mature adenovirus virion leads to
the formation of intermediate capsids, some of which
may contain little viral or cellular DNA [11-13]. Earlier,
we demonstrated that viral RNAs were packaged in por-
cine adenovirus (PAdV) -3 virions, a non-enveloped DNA
Published: 5 February 2009
Virology Journal 2009, 6:16 doi:10.1186/1743-422X-6-16
Received: 22 December 2008
Accepted: 5 February 2009
This article is available from: />© 2009 Xing and Tikoo; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Virology Journal 2009, 6:16 />Page 2 of 7
(page number not for citation purposes)
virus [14]. Another report suggested that a non-viral RNA
(LacZ mRNA) transcribed from a recombinant human
adenovirus (HAdV) -5 is packaged into the HAdV-5 viri-
ons [15]. In this report, we examined the incorporation of
viral RNAs in mature and empty/intermediate capsids of
PAdV-3 and HAdV-5. Moreover, the characteristics of
packaged viral RNAs were further examined by Southern
blot hybridization and real-time RT-PCR analysis.
Methods
Cells and Viruses
VIDO R1 cells [16] were grown in Eagle's minimum essen-
tial medium supplemented with 10% heat inactivated
fetal bovine serum (FBS). The wild-type (wt) PAdV-3
(6618 strain) [17] and mutant Pav3-PL1 [18] were propa-
gated and titrated in VIDO R1 cells. 293 cells [19] were
grown in Dulbecco's modified Eagle's medium supple-

mented with 5% FBS. The wild-type (wt) HAdV-5 and
recombinant HAV5.EGFP containing enhanced green flu-
orescent protein (EGFP) gene inserted in E1 region of
HAdV-5 were propagated and titrated in 293 cells.
Isolation of viral capsids
In order to obtain empty/intermediate and mature cap-
sids, VIDO R1 cells were infected with wt PAdV-3 or
mutant Pav3-PL1 at a multiplicity of infection (MOI) of
10 plaque forming units (PFUs). At 48 h post infection,
the infected cells were collected and freezed-thawed three
times. The cell lysates were subjected to a step gradient
with 1.2 and 1.4 g of CsCl/ml and ultracentrifuged at
35,000 rpm for 2 h. Two major bands were harvested and
loaded on a continuous CsCl gradient at 1.32 g/ml and
centrifuged at 35,000 rpm overnight. The bands from this
gradient were further purified on a third gradient and then
dialyzed into phosphate-buffered saline (PBS). Similarly,
HAdV-5 capsids were purified from 293 cells infected with
wt HAdV-5 or recombinant HAV5.EGFP. Total RNA was
isolated from purified capsids, or virus infected cells as
described earlier [14].
Electron microscopy
CsCl-purified virions were adsorbed to nickel grids. After
adsorption, the grids were stained with 1% solution of
phosphotungstic acid for 60 s and examined by using
transmission electron microscope (Philips EM410). Pho-
tographs were taken from representative areas from each
sample.
Southern blot
The wt PAdV-3 genomic DNA was isolated from purified

viral capsids as described earlier [14,18]. Viral genomic
DNA or pFHAV5 plasmid DNA containing E1A-deleted
HAdV-5 genome was digested with HindIII and loaded
into 1% agarose gel. Fractionated DNAs were transferred
into Gene Screen Plus hybridization transfer membranes
(PerkinElmer) and probed with [
32
P]-labeled cDNA
probes prepared by reverse transcribing 2 μg of RNAs iso-
lated from purified capsids or virus infected cells in the
presence of [
32
P]-labeled dCTP as described earlier [14].
Real-time PCR
Real-time PCR was performed with Platinum Quantitative
PCR SuperMix-UDG kit (Invitrogen) as instructed by
manufacturer. The cDNAs were prepared by reverse tran-
scription as described previously [14]. The synthesized
cDNAs or viral genomic DNAs were used as templates in
real-time PCR, using iCycler real-time PCR system (Bio-
Rad). TaqMan probes for E1B
small
(5'-TTTATC AAGGTAG-
TAGCAG AGGCCA-3') and fiber (5'-TCCCTGGGTCCCG-
GTCT TTCTA ACT-3') were labeled with FAM at 5'-end
and TAMRA at 3'-end, and purchased from Qiagen. The
primers RTE1B1 (5'-AG TACA GGGGT CTCAGAACT-3')
and RTE1B2 (5'-CTCCACAA A A ATCTCAATCA-3') are
specific for PAdV-3 E1B
small

, TRFIB1 (5'-GAT-
GGCAAGCTGG TTCTC A A-3') and TRFIB2 (5'-GGAGCT-
GTGACTTGCAGA CT-3') are specific for PAdV-3 fiber
[20]. After incubation at 50°C for 2 min and 95°C for 2
min, the reaction was run for 45 cycles with denaturation
at 95°C for 15 s, annealing and extension at 60°C for 30
s. As a negative control, RNase-treated RNA samples were
also reversely transcribed and simultaneously subjected to
PCR reaction. Serial 10-fold dilutions of known amount
of plasmid pFPAV3 DNA (known as pPAV200)[21] con-
taining E1B
small
and fiber genes were used as positive con-
trol to generate the standard curve. The copy numbers
were calculated by converting the weight unit of double-
stranded pFPAV3 DNA into the numbers of single-
stranded DNA molecules. The averaged cycle threshold
(Ct) values of five replicates were used to determine the
relative RNA copy number.
Results and discussion
Preparation and characterization of PAdV-3 capsids
At late times during adenovirus infection, two kinds of
particles are abundant, which can be separated by CsCl
gradient centrifugation [22]. The heavy particles exhibit-
ing a density of approximately 1.32 g/ml are the mature
capsids that are composed of the capsid proteins, the core
proteins and the viral DNA. The lighter particles exhibit-
ing a density of approximately 1.29 g/ml are the empty/
intermediate capsids that are composed of the capsid pro-
teins, but are usually devoid of the genomic DNA and the

core proteins. In order to obtain different capsids, we
chose to purify mutant Pav3-PL1, (reduced DNA packag-
ing efficiency) [18] and wt PAdV-3. As seen in Fig. 1A, two
major bands and a minor band were observed in wt PAdV-
3 or in mutant Pav-3-PL1 infected cell lysate after CsCl
gradient centrifugation. As expected, the upper band was
heavier than the lower band in Pav-3-PL1 infected cells.
The yields of both empty/intermediate and mature cap-
Virology Journal 2009, 6:16 />Page 3 of 7
(page number not for citation purposes)
sids were quantified by measuring protein concentration
with Bradford protein assay (Bio-Rad).
To further characterize the integrity of empty/intermedi-
ate and mature capsids, DNA was isolated from both
bands. Ethidium bromide-stained agarose gel showed
that the viral genomic DNA is present predominantly in
the lower bands (data not shown), confirming that the
viral particles with the density of 1.32 g/ml are the mature
capsids. To determine the integrity of the capsids, trans-
mission electron microscopy was performed as described
[23]. Analysis of the preparations by the electron micro-
scope (EM) suggested that the mature and the empty/
intermediate capsids of both wt PAdV-3 and mutant Pav3-
PL1 are predominantly intact (Fig 1B, wild-type PAdV-3).
Viral RNA was mainly detected in mature capsids and
appeared polyadenylated
Earlier studies demonstrated that most of the viral RNAs
were nonspecifically packaged in HCMV and KSHV parti-
cles [1,3,6]. We reasoned that if the viral RNAs were non-
specifically incorporated into PAdV-3 particles as well, the

empty/intermediate capsids would be expected to contain
more viral RNAs than the full capsids, since the absence of
viral genome in the empty/intermediate capsids would
leave extra room for RNAs. To test this hypothesis, equal
amounts (based on protein concentrations) of CsCl puri-
fied empty/intermediate and mature capsids of wt PAdV-
3 were treated with RNase to remove RNAs contaminated
outside of viral particles before processing for the isola-
tion of virion RNAs as previously described [14]. The iso-
lated virion RNAs were treated with RNase-free DNase
(Ambion) to eliminate the contaminated viral genomic
DNA, followed by addition of 0.1 volume DNase inactiva-
tion reagent (DNA-free kit, Ambion). The RNA was also
isolated from wt PAdV-3 infected cells as described previ-
ously [14]. Two micrograms of virion RNAs from each
preparation were converted into [
32
P]-labeled cDNAs by
reverse transcriptase II (Invitrogen) with oligo-dT and
hexamers in the presence of [
32
P]-dCTP as described pre-
viously [14]. As a control, two microgram of virion RNAs
from each preparation were treated with RNase before RT
reaction. The resultant cDNAs were subsequently hybrid-
ized to the membrane blots containing HindIII-digested
wt PAdV-3 genomic DNA. Positive signals were detected
with radioactive probes made from the RNAs isolated
from wt PAdV-3 infected cells (Fig. 2, panel a). Similarly,
positive signals were also detected with radioactive probes

made from the virion RNA isolated from the mature cap-
sids (Fig. 2, panel d). No signal could be detected with
radioactive probes made from the RNase treated RNAs
isolated from PAdV-3 infected cells (Fig. 2, panel a) or
purified mature capsids (Fig 2, panel d) indicating that the
Production of the virus capsidsFigure 1
Production of the virus capsids. (A) Crude lysates pre-
pared from VIDO R1 cells infected with Pav3-PL1 or wt
PAdV-3 were separated by centrifugation through CsCl gra-
dients. The position of empty/intermediate and mature cap-
sids is indicated. (B) Electron microscopic (EM) images of
CsCl-purified viral capsids.
A)
Mature
Empty/Intermediate
Mutant WT
B)
100nm
Mature capsid
Empty / intermediate capsid
100nm
Southern hybridization with PAdV-3 DNAFigure 2
Southern hybridization with PAdV-3 DNA. Fraction-
ated HindIII digested wt PAdV-3 genomic DNA was probed
with [
32
P]-labled cDNAs generated by RT of the RNAs iso-
lated from purified wt PAdV-3 mature capsids, empty/inter-
mediate capsids, wt PAdV-3-infected VIDO R1 cells or
uninfected ST cells as described in the text. Blots in c, d, e,

and f were exposed for the same time with PhosphorImager
screen (Bio-Rad). Presence (+). Absence (-). EtBr, Ethidium-
bromide (EtBr) staining of HindIII digested PAdV-3 genomic
fragments.1 Kb DNA ladder (M). Mature(Ma);Empty/Inter-
mediate(E-I).
Virus-infected
VIDO R1 cell
Hexamers
Oligo (dT)
DNase
RNase
EtBr
a
M
b
cde
+
++ + +
++
+
+
í
++ ++
+++
RNA
í
+
í + í +
í
++í +

í
+
+
+
+++
+
+
+
+
+
+
íí
+
íí
+
+
M
f
Ma MaST cell E - I
Virology Journal 2009, 6:16 />Page 4 of 7
(page number not for citation purposes)
positive signals were detected due to labeled probes gen-
erated from the viral RNAs. These results confirmed our
earlier observation that the viral RNAs are packaged in the
mature capsid [14]. Compared with mature capsids (Fig.
2, panel d), a weak signal was detected with radioactive
probes made from the RNAs isolated from the empty/
intermediate capsids (Fig. 2, panel e), suggesting that
empty/intermediate capsids might contain only trace
amounts of viral RNAs. The trace virion RNAs detected in

the empty/intermediate capsids might be the RNAs pack-
aged nonspecifically in empty/intermediate capsids.
Taken together, the results indicated that the viral RNAs
were mainly packaged in the mature capsids containing
viral genomic DNAs.
To further characterize the viral RNAs packaged in PAdV-
3 virions, reverse transcription (RT) of the RNAs from the
mature capsids was primed with only oligo-dT of 12–16
nucleotides (nt), which is specific for mRNAs containing
polyA tail. As a positive control, 2 μg of total RNA isolated
from wt PAdV-3-infected VIDO R1 cells at late times post
infection was reversely transcribed in the presence of
[
32
P]-dCTP as described above. As a negative control, 2 μg
of total RNA from swine testis (ST) cells was reversely tran-
scribed in the presence of [
32
P]-dCTP. As expected, radi-
olabeled probes made from the RNAs isolated from
uninfected ST cells did not produce any positive signals in
Southern blot (Fig. 2, panel c). In contrast, radiolabeled
probes made from RNAs isolated from wt PAdV-3-
infected VIDO R1 cells resulted in strong signals, indicat-
ing that PAdV-3 specific RNAs were present in cellular
RNA preparations (Fig. 2, panel b). No signal could be
detected with radiolabeled probes made from the RNase
treated RNA isolated from wt PAdV-3-infected VIDO R1
cells (Fig. 2, panel b). Compared with the oligo-dT and
hexamer-doubly primed RT reaction (Fig. 2, panel a), the

oligo-dT primed RT reaction displayed weaker positive
signals (Fig. 2, panel b). Compared to the results obtained
with total RNAs from the virus infected VIDO R1 cells, the
oligo-dT primed RT of virion RNAs from purified mature
capsids also produced weaker positive signals (Fig. 1B.
panel f) than RT primed with both oligo-dT and hexamer
(Fig. 2. panel d). These results suggested that some viral
RNAs packaged in the virions appear to be polyade-
nylated.
Viral RNA was packaged at a low copy number into PAdV-
3 mature capsids
To quantify the RNAs packaged in the virions, we per-
formed real-time RT-PCR with Platinum Quantitative
PCR SuperMix-UDG kit (Invitrogen). We choose to quan-
titate RNAs specific for an early region gene (E1B
small
) and
a late region gene (fiber) of PAdV-3. The viral genomic
DNA and RNA were isolated from the same pool of puri-
fied mature capsids. Viral DNA was used as a template in
real-time PCR to determine the total number of viral par-
ticles containing viral genome. The isolated RNAs with or
without RNase treatment were reversely transcribed with
the oligo-dT and hexamer as described above before using
as templates in the real-time PCR to determine the RNA
copy number based on the cycle threshold (Ct) values.
Real-time PCR was performed by using Cycler real-time
PCR system (Bio-Rad) as described in Methods.
The RNA copy number was finally normalized with the
total number of genome-containing particles designated

as 100%, and shown as relative percentage. The copy
number of E1B
small
and fiber RNAs in mature capsids were
5% and 15% respectively (Fig. 3) of the number of
genome-containing particles, indicating that no more
than one copy of E1B
small
or fiber RNA was packaged in a
mature capsid. Earlier report also suggested that at the
most one copy of a reporter mRNA produced from recom-
binant HAdV-5 was packaged in the viral particles [15].
Human adenovirus type 5 particles also contain viral RNAs
To determine if viral RNAs were packaged into HAdV-5
virions, both empty/intermediate and mature capsids
Quantitation of viral RNAs by real-time PCRFigure 3
Quantitation of viral RNAs by real-time PCR. Viral
genomic DNA and RNAs were isolated from the same pool
of purified mature capsids. RNAs with (+) or without (-)
RNase treatment were reversely transcribed with oligo-dT
and hexamers, and then used as templates in the real-time
PCR with TaqMan probes. The calculated copy number of
the mRNAs of E1B
small
and fiber is shown as percentage rela-
tive to the copy number of the viral genomic DNA. The
averaged cycle threshold (Ct) values of 5 replicates were
used to determine the relative RNA copy number.
0
20

40
60
80
100
120
RNase
í + í +
Genome
R
e
l
a
t
i
v
e
c
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R
N
A
s
i
n
m
a
t
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e
c
a
p
s
i
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(
%
)
E1B
Fiber
Virology Journal 2009, 6:16 />Page 5 of 7
(page number not for citation purposes)
were purified from wt HAdV-5 infected 293 cells [19]
using the procedure described earlier [14]. Equal amounts

(based on protein concentrations) of CsCl purified HAdV-
5 capsids were treated with RNase and then extracted with
Trizol reagent (Invitrogen) to isolate the virion RNAs. Two
micrograms of virion RNAs isolated from mature or
empty/intermediate capsids were reversely transcribed by
reverse transcriptase II in the presence of [
32
P]-dCTP. The
synthesized [
32
P]-labeled cDNAs were hybridized to Hin-
dIII-digested plasmid pFHAV5 that contains E1A-deleted
HAdV-5 genome. Positive signals were detected with radi-
oactive probes made from the RNAs isolated from wt
HAdV-5 infected 293 cells (Fig. 4, panel e). Similarly, pos-
itive signals were also detected with radioactive probes
made from the virion RNA isolated from mature capsids
(Fig. 4, panels a, c, and d). No signal could be detected
with radioactive probes made from the RNase treated
RNAs isolated from wt HAdV-5 infected 293 cells (Fig. 4,
panel e) or mature capsids (Fig. 4, panels a, c, and d), indi-
cating that positive signals were detected due to labeled
probes generated from viral RNAs. Similar to wt PAdV-3
(Fig. 2, panel b), a weak signal was detected with radioac-
tive probes made from the RNA isolated from empty/
intermediate capsids (Fig. 4, panel b). The oligo-dT/hex-
amer (Fig 4, panel a), hexamers alone (Fig. 4, panel c),
and the oligo-dT alone (Fig. 4, panel d)-primed RT of the
virion RNAs from mature capsids all resulted in positive
signals in Southern blot analysis. The data indicated that

wt HAdV-5 particles containing the genomic DNA also
contain more viral RNA and that part of the virion RNAs
are potentially polyadenylated. The phenomenon of
packaging of viral RNAs in adenoviral particles is not
unique to PAdV-3 that naturally infects pigs.
Viral RNA packaging appears to be associated with
adenovirus genomic DNA encapsidation
To determine why the viral RNAs are predominantly
detected in adenoviral mature capsids, the RNA and DNA
were quantitatively analyzed in CsCl purified mature and
empty/intermediate capsids of HAV5.EGFP. CsCl purified
mature and empty/intermediate HAV5.EGFP capsids were
mock treated or treated with RNase A (Ambion) and
DNase I (Invitrogen) at room temperature. After 4 h incu-
bation at room temperature, 10 μl of each sample was dis-
solved in RIPA buffer [16,21]. Proteins from capsid lysates
were separated by sodium dodecyl sulphate – polyacryla-
mide gel electrophoresis (SDS-PAGE) and stained with
SYPRO Ruby protein stains (Bio-Rad) overnight. Quanti-
tative analysis of adenoviral hexon protein (Fig. 5A) sug-
gested that the RNase/DNase treatment did not affect the
virion protein concentration.
Since both mature and empty/intermediate capsids may
contain viral/cellular DNA [11-13], initially we quanti-
tated the viral DNA in these capsids. Equal amounts
(based on protein concentrations) of CsCl purified cap-
sids were treated with RNase A and DNaseI as described
above. The samples were further treated with 100 μg/ml
proteinase K (Invitrogen) at 50°C for 2 h, before extract-
ing sequentially with phenol and chloroform. The DNA

was precipitated by isopropanol and dissolved in RNase A
containing TE buffer (10 mM Tris-Cl, 1 mM EDTA,
pH8.0). Quantitative analysis suggested that mature cap-
sids yielded 100 times more DNA than empty/intermedi-
ate capsids irrespective of the RNase/DNase treatment
(Fig. 5B). The significant difference in the amount of DNA
obtained from mature and empty/intermediate capsids is
consistent with the general suggestion that mature capsid
contains mature viral genomic DNA and empty/interme-
diate capsid may contain little premature viral DNA [11-
13]. Moreover, DNA in mature and empty/intermediate
capsids is not accessible to DNase/RNase treatment of the
intact capsids. To confirm this further, we examined the
expression of EGFP in 293 cells infected with equal
amounts of mature or empty/intermediate capsid samples
with or without RNase/DNase treatment. As seen in Fig.
5C, EGFP expression was detected in cells infected with
mature or empty/intermediate capsids. However, empty/
intermediate capsids transduced 10
4
fold less cells than
the mature capsids. These results suggested that empty/
intermediate capsids may contain premature viral and/or
cellular DNA. Earlier reports have suggested that some of
the intermediate capsids may contain left end of the ade-
novirus genome [11,12].
Next, we quantified the RNAs present in the capsids. RNA
was extracted from equal amounts (based on protein con-
centrations) of CsCl purified HAV5. EGFP mature and
empty/intermediate capsids as previously described [14].

Equal amount (42 μg) of total RNAs were isolated from
Southern hybridization with HAdV-5 DNAFigure 4
Southern hybridization with HAdV-5 DNA. Fraction-
ated HindIII digested pFHAV5 DNA was probed with [
32
P]-
labeled cDNAs generated by reverse transcription of RNAs
isolated from purified wt HAdV-5 mature capsids, empty/
intermediate capsids, or wt HAdV-5-infected 293 cells. Blots
in a, b, c, d, and e were exposed for the same time with
PhosphorImager screen (Bio-Rad). Presence (+). Absence (-).
Ethidium-bromide (EtBr) staining of HindIII digested pFHAV5
DNA fragments. 1 Kb DNA ladder (M). DNA band sizes are
indicated in bps on the right. Mature(Ma); Empty/Intermedi-
ate(E-I).
1650
2000
3000
5000
12000
Hexamers
Oligo (dT)
DNase
RNase
EtBr
Ma
a
í
+
í +

í +
í ++
í
M
E - I Ma Ma
Virus infected
293 cell
b
cde
+
+
++
++
++ +
+
+
+
++
++
í ++
+
í
++ ++ ++
+++
RNA
Virology Journal 2009, 6:16 />Page 6 of 7
(page number not for citation purposes)
both mature and empty/intermediate capsids without
RNase/DNase treatment (Fig. 6A). However, RNase/
DNase treatment reduced the total RNA yields (only 2 μg)

of both mature or empty/intermediate capsids by 21 fold
(Fig. 6A). These results indicated that a large amount of
RNAs bound to the capsid surface could not be removed
by CsCl ultracentrifugation. To analyze the amount of
viral RNAs present in the total RNAs isolated from mature
and empty/intermediate capsids, 2 μg of RNA isolated
from mature or empty/intermediate capsids without
RNase/DNase treatment was reversely transcribed by
reverse transcriptase II (Invitrogen) in the presence of
[
32
P]-dCTP. Synthesized [
32
P]-labeled cDNAs were
hybridized to HindIII-digested plasmid pFHAV5 contain-
ing E1A-deleted HAdV-5 genome. As expected, the cDNA
probes resulted in positive signals as strong as the cDNA
probes made from wt HAdV-5 infected 293 cell RNA (Fig.
6B). This is consistent with the suggestion that CsCl puri-
fication does not remove the RNAs bound to capsid sur-
faces.
To determine the identity of the RNAs isolated from
RNase/DNase treated mature and empty/intermediate
capsids, cDNA probes were synthesized using 2 μg of RNA
isolated from mature or empty/intermediate capsids with
RNase/DNase treatment and hybridized to HindIII-
digested plasmid pFHAV5 containing E1A-deleted HAdV-
5 genome. As seen in (Fig. 6B), these cDNA probes also
resulted in positive signals, confirming that there are viral
RNAs present inside the capsids. Although the total RNA

yields from mature and empty/intermediate cpasids with
RNase/DNase treatment appeared similar (Fig. 6A), the
quantitative analysis indicated that the mature capsids
contain more (30 fold) viral RNAs than empty/intermedi-
ate capsids (Fig. 6C). It is possible that the empty/interme-
diate capsids contain more cellular RNA. However,
absence of detection of cellular mRNAs of house-keeping
genes such as actin in virion RNAs [14] raises the possibil-
ity that the majority of the cellular RNAs isolated from
mature and empty capsids might be rRNAs.
Although the virion RNAs were detected in the mature
capsids, the low copy number of examined RNAs suggests
that the viral RNAs might be packaged in a passive way by
accompanying the viral genomic DNA at the stage of DNA
Analysis of viral protein and DNA in HAV5.EGFP capsidsFigure 5
Analysis of viral protein and DNA in HAV5.EGFP
capsids. (A) Proteins from lysates of RNase/DNase treated
or untreated mature and empty/intermediate capsids were
separated by 10% SDS-PAGE, and stained by SYPRO Ruby
protein stains. A 100 kDa hexon protein band was used for
determining the capsid protein concentration using Kodak IM
Network software. (B) Total yields of DNAs isolated from
mature and empty/intermediate capsids with or without
RNase/DNase treatment. (C) EGFP expression. 293 cells
were transduced by 10 fold serially diluted mature and
empty/intermediate capsids with or without RNase/DNase
treatment. At 48 h post transduction, cells were analysed for
EGFP expression by a fluorescent microscope (i) EGFP, (ii)
Phase contrast. Presence (+). Absence (-). Mature (Ma);
Empty/Intermediate (E-I).

Viral DNA
(μg)
í
+
í
+
Capsid
E - I
Ma
DNase
RNase
í
+
+
í
0
500
1000
1500
2000
2500
3000
3500
4000
Viral Protein
(A)
(B)
12
3
4

0
50
100
150
200
250
300
0
1
2
3
12
3
4
3
4
Relative intensity
E - I
MA
Capsid
DNase
RNase
í
++
í
í
+
í
+
i

ii
(C)
Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatmentFigure 6
Analysis of RNA in HAV5.EGFP capsids with or with-
out RNase/DNase treatment. (A) Total yields of RNAs
isolated from mature and empty/intermediate capsids with or
without RNase/DNase treatment. (B) The [
32
P]-labeled
cDNAs were made by reverse transcription of 2 μg, RNase-
free DNase treated RNAs from mature and empty/interme-
diate capsids with or without RNase/DNase treatment and
hybridized to HindIII-digested pFHAV5, which contains E1A-
deleted HAdV-5 genome. RT was primed by oligo-dT/hex-
amers.(C) Viral RNAs detected in RNAs from mature (Ma)
and empty/intermediate (E-I) capsids after RNase/DNase
treatment in Southern hybridization in panel B were quanti-
tated by using PhosphorImager software.
0
5
10
15
20
25
30
35
40
45
50
Total RNA

(μg)
(A)
1
2
3
4
Viral RNA
(C)
0
5000
10000
15000
20000
25000
30000
35000
40000
1
2
3
4
Relative intensity
í
+
í
+
Capsid
E - I
Ma
DNase

RNase
í
+
+
í
Hexamers
Oligo (dT)
DNase
RNase
í
+
í +
í +
í +
E - IMa
+
++
+
++
++
+
++ +
++
++
+
++ ++ ++
+
RNA
Capsid
DNase

RNase
í +
+
í
í +
+
í
(B)
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Virology Journal 2009, 6:16 />Page 7 of 7
(page number not for citation purposes)
encapsidation. So far, two models for adenovirus assem-
bly have been proposed on the basis of pulse-chase anal-
ysis and the study of mutant viruses. The traditional
model is that the adenovirus assembly begins with the for-
mation of empty capsids, followed by the insertion of
viral DNA into preformed empty capsids [24-26]. How-
ever, recently proposed model suggests that the capsids
are assembled around the viral genomic DNA [27,28]. The

insertion of viral genome into the preformed empty cap-
sids more likely would separate the synthesized RNAs
from viral genome, resulting in missing of the viral RNAs
in full capsids containing the viral genome. Thus, a mech-
anism of adenovirus assembly where packaging of virion
RNAs with genomic DNA is coordinated with capsid for-
mation appears to be plausible.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LX designed and carried out the experiments, and helped
to analyze the data and draft the manuscript. SKT helped
to design the study, help in interpretation of the data and
edit the manuscript. Both authors read, made corrections
and approved the final manuscript.
Acknowledgements
This work was supported by the grants from Natural Sciences and Engi-
neering Research Council (NSERC) of Canada to SKT Published as VIDO
article # 455.
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